Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2570-2575, March 1996 Biochemistry Inhibition of the association of RNA polymerase II with the preinitiation complex by a viral transcriptional repressor (transcription/repression/immediate early protein 2/human cytomegalovirus) GARY LEE*, JUN WUtt, PERCY Luu*, PETER GHAZALt, AND OSVALDO FLORES*§ *Tularik Inc., South San Francisco, CA 94080; and tDepartments of Immunology and Neuropharmacology, Division of Virology, The Scripps Research Institute, La Jolla, CA 92037 Communicated by Robert Tjian, University of California, Berkeley, CA, October 17, 1995 ABSTRACT Transcriptional repression is an important start site of the HCMV MIEP (7-9). More recently, it was component of regulatory networks that govern gene expres- shown that purified IE2 protein binds directly to the crs sion. In this report, we have characterized the mechanisms by element (10, 11) and represses transcription of the HCMV which the immediate early protein 2 (IE2 or IE86), a master MIEP in vitro (12, 13). In this work, we have employed a transcriptional regulator of human cytomegalovirus, down- purified in vitro transcription system to study the precise regulates its own expression. In vitro transcription and DNA mechanism by which IE2 represses transcription. Our obser- binding experiments demonstrate that IE2 blocks specifically vations show that IE2 does not impede promoter recognition the association of RNA polymerase II with the preinitiation by TFIID, yet very effectively blocks recruitment of RNA complex. Although, to our knowledge, this is the first report polymerase II. to describe a eukaryotic transcriptional repressor that selec- tively impedes RNA polymerase II recruitment, we present MATERIALS AND METHODS data that suggests that this type ofrepression might be widely used in the control of transcription by RNA polymerase II. Purification of RNA Polymerase II and General Factors. The RNA polymerase II fractions used in the in vitro tran- Substantial progress has been made in the identification and scriptions (DEAE 5PW step) and in the DNA binding exper- functional characterization of eukaryotic transcriptional acti- iments (Ila form, alkyl-Superose step) were purified as de- vators (for review, see ref. 1). In contrast, the understanding scribed (14). TFIIA and TFIID were purified as described (15). of transcriptional repressors in eukaryotes has received con- TFIIF, TFIIH, recombinant TBP, IIB, IIEp34, and IIEp56 siderably less attention. Studies on transcriptional repression proteins were purified as described (16-20). in prokaryotes have led to the hypothesis that eukaryotic Expression and Purification of Recombinant IE2 Proteins. repressors might function by a competitive mechanism, in Both the full-length IE2 cDNA and a DNA fragment encoding which the DNA-bound repressor blocks access of the basal IE2p40 were cloned into a pET-15b vector (Novagen). The transcription machinery to promoter DNA. Perhaps for this DNA fragment encoding IE2p4O was isolated with the use of reason, one of the first eukaryotic repressors to be recognized the polymerase chain reaction (PCR). The conditions used to was the simian virus 40 tumor antigen, which represses tran- express and purify the full-length IE2 protein and the IE2p40 scription by masking the promoter and inhibiting its recogni- protein were identical. Briefly, cultures (BL21) were grown to tion by the general transcription machinery (2, 3). The precise an OD600 of 0.5-1.0 unit and induced with 0.5 mM isopropyl step at which preinitiation complex formation is blocked by the j3-D-thiogalactoside (BRL) for 2 h at 37°C. Bacterial extracts simian virus 40 tumor antigen is unknown. The Drosophila were prepared, and the proteins were purified according to the P-element transposase (4), the cellular DNA binding protein conditions described in the Novagen His-Bind buffer kit LBP-1 (5), and the bovine papilloma virus E2 protein (6) protocols. The affinity-purified proteins were then fraction- represent additional examples of repressors that block binding ated on a Mono Q column (HR5/5, Pharmacia). The molar ofthe preinitiation complex to DNA. In each case, it was found concentration was calculated by assuming that these proteins that the repressor blocked basal transcription factor (TF) IID exist as dimers in solution. binding to the TATA box, suggesting that the first step in In Vitro Transcription Reactions. Transcription reaction preinitiation complex formation was the target for inhibition mixtures contained TFIID (DE-52, 1.0 ,ug) or recombinant by this class of eukaryotic repressors. human TATA binding protein (TBP) (60 ng), recombinant Important contributions to all aspects of eukaryotic gene human TFIIB (20 ng), recombinant human TFIIE (100 ng), regulation have come from studies aimed at understanding the TFIIF (TSK phenyl, 200 ng), TFIIH (400 ng), RNA polymer- coordinated regulation ofviral gene expression. Excellent viral ase II (DEAE 5PW, 100 ng), and TFIIA (DE-52, 5 jig). TFIIA model systems include the dual-function activator/repressor was not used in reactions reconstituted with TBP. Reaction proteins encoded by herpesviruses. Human cytomegalovirus conditions were as described (17). Reaction mixtures were (HCMV) is a 13-herpesvirus that has become a common and incubated at 30°C for 45 min or as indicated. very serious pathogen in immunocompromised patients. In Gel Mobility Shift Assay. The protein components indicated this report, we have studied the mechanism by which the in the figure legend were incubated with a 3'-end-labeled DNA HCMV immediate early protein 2 (IE2) represses transcrip- fragment (80 bp) containing the core promoter sequences of tion of its own promoter, the potent major immediate early the HCMV MIEP (bp -34 to +5) and polylinker sequences of enhancer-promoter (MIEP) of HCMV. Transient transfection the plasmid Bluescript SKI (Stratagene). Reaction conditions experiments indicate that repression by IE2 requires a cis- regulatory DNA element referred to as cis-repression signal Abbreviations: IE, immediate early protein; HCMV, human cytomeg- (crs) located between the "TATA" box and the transcription alovirus; MIEP, major immediate early enhancer-promoter; crs, cis- repression signal; TF, transcription factor; TBP, TATA binding pro- tein; TAF, TBP associated factor. The publication costs of this article were defrayed in part by page charge lPresent address: Signal Pharmaceuticals Inc., 555 Oberlin Drive, payment. This article must therefore be hereby marked "advertisement" in Suite 100, San Diego, CA 92121. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 2570 Downloaded by guest on September 29, 2021 Biochemistry: Lee et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2571 were as described (16) and TBP (3 ng), IIB (1 ng), IIF (20 ng), works effectively with TBP alone, we do not believe that IE2 RNA polymerase Ila (alkyl-Superose fraction, 10 ng), and functions by attenuating a TAF-mediated activation step. This IE2p4O (2 fmol) were added as indicated in Fig. 6. The reaction interpretation is consistent with the fact that the assay we have products were separated by electrophoresis on a 4% polyacryl- utilized represents a minimal activator-independent reaction. amide gel [30% (wt/vol) acrylamide/0.8% N,N'-methylene The experiments described above show that we can recapitu- bisacrylamide] containing 2.5% (vol/vol) glycerol and lx TBE. late transcriptional repression by IE2 with the use of a purified in vitro transcription system. Repression by IE2 Is Dependent on the Position of the crs RESULTS Element. Because the crs element is located between the IE2 Represses the HCMV MIEP in a Transcription System TATA box and the transcription start site, it is plausible that Reconstituted with Purified Factors. To characterize the the mechanism of repression by IE2 might involve steric mechanism of repression by IE2, we developed a purified in hindrance with the basal transcription complex. To investigate vitro transcription system that supported accurate transcription this possibility we asked whether the position of the crs initiation from the HCMV MIEP. Simultaneous addition of element with respect to the TATA box was important for IE2 resulted in repression. We analyzed the effect of IE2 on HCMV MIEP basal factors with recombinant full-length constructs in which the distance between the crs element and significant transcriptional repression of reactions driven by the the TATA box was increased by 5, 10, or 15 bp (Fig. 2). To wild-type HCMV MIEP (Fig. 1B, lanes 1-10). The level of analyze these promoter mutants, we employed run-off assays. repression increased in proportion to the amount of IE2 As shown in Fig. 2, IE2 retained the ability to repress tran- protein added. In contrast, IE2 had little effect on transcrip- scription when the distance between the crs element and the tion reactions containing a mutant HCMV MIEP with four TATA box of the HCMV MIEP was increased from 10 to 15 nucleotide substitutions in the crs element (Fig. 1 A and B, nt (TATA 5 crs), indicating that IE2 does not require a specific lanes 11-15). These observations are consistent with previous stereo alignment with the TATA box to effect repression. In studies indicating that repression by IE2 requires direct bind- contrast, the ability of IE2 to repress transcription was dra- ing of IE2 to the crs element (11, 12). Quantitation of this matically decreased when the crs element was positioned 20 or experiment indicated